The thymus is a specialized organ for the development of T cells. Within the thymus, cortical epithelial cells play a crucial role in the generation of T cells with appropriate specificity and function. In this project, we will study the role of a gene expressed uniquely in cortical epithelial cells, 25t, which is involved in proteolysis and is required for positive selection of CD8 T cells. We will further define and study key cortical epithelial cell identify genes by creating reporter mice to facilitate the purification or depletion of these cells in vivo. We will also determine if 25T and other genes are altered in situations where T cell development is impaired, and determine if manipulation of these genes can be used to support T cell reconstitution in vitro. Another focus of this proposal is to define the genetic changes required for the functional maturation that occurs after positive selection. Specifically, we will determine: what gene changes occur at the semi-mature to mature SP transition, which changes are critical for development of proliferation competence, and if specific stromal cell interactions are required for competence PUBLIC HEALTH RELEVANCE: The thymus is a specialized organ for the development of T cells. Within this organ, cortical epithelial cells play a crucial role in the generation of T cells with appropriate specificity and function. This application proposes to explore the molecular mechanisms by which cortical epithelial cells support T cell differentiation, and to exploit this knowledge in developing an in vitro T cell reconstitution system that can be used for therapeutic purposes.